ch 20 Biotechnology

stem cell

undifferentiated cells stem cells in embyos are pluripotent: can differentiate into many cell types in adults: cam produce only a few cell types

GM plants

use bacteria to get foreign genes into plant cells carry genes for resistance to herbicide, pest, frost, drought resistance add in vitamins

gel electrophoresis

A technique used for separating nucleic acids or proteins on the basis of their size and electrical carge, both of which affect their rate of movement through an electric field in a gel. Gel is made of a polyemer such as agarose. Each sample, a mixture of DNA molecules, is placed in a separate well near one end of the agarose gel. The gels is set into a small plastic support and immersed in an aqueous, buffered solution in a tray with electrodes at each end. When the current is turned on, DNA moves from negative to positive electrode. Larger fragments move slower than shorter molecules Used in DNA fingerprinting

insertion in vector

Both DNA samples are cut with the same restriction enzyme Fragments are mixed together, allowing base pairing between their complementary sticky ends. DNA ligase is added, which covalently bonds the sugar-phosphate backbones of the fragments whose sticky ends have base paired. The products include recombinant plasmids and many nonrecombinant plasmids

reinfect cells

DNA mixture is added to bacteria that have mutation in the lacZ gene (codes for enzymes that break down lactose) making them unable to hydrolyze lactose. The cells take up foreign DNA by transformation. Some cells acquire a recombinant plasmid carrying a gene while others take up a nonrecombinant plasmid.

recombinant DNA

DNA molecules formed from different sources

problems with expressing cloned eukaryotic genes in bacteria

Differences in promoters (fixed through expression vectors) Introns prevent correct expression of the gene by bacterial cells because they do not have RNA splicing machinery (fixed through cDNA's)

DNA sequencing

Fragment of DNA to be sequenced is denatured into single strands and incubated in a test tube with a primer designed to base-pair with the known 3' end of the template strand, DNA polymerase, the four deoxyribonucleotides, four dedoxyribonuclotides, each tagged with a specific fluorescent molecule. Synthesis of each new strand starts at the 3' end of the primer and continues until a dideoxyribonucleotide is inserted, at random, instead of the normal equivalent deoxyribonucleotide. This prevents further elongation of the strand. A set of labeled strands of various lengths is generated, with the colo of the tag representing the last nucleotide in the sequence. The labeled strands in the mixture are separated by passage through a polyacrylamide gel, with shorter strands moving throuh more quickly. For DNA sequencing, the gel is formed in a capillary tube . The small size of the tube allos a fluorescence detector to sense the color of each fluorescent tag as the strands come throgh. Strands differeing in length by as little as one nucleotide can be distinguished from each other.

polymerase chain reaction (PCR)

amplifies DNA (makes more copies) done in vitro goes through 3 same cycles Denaturation: reaction mixture is heated to denature (separate) the DNA strands Annealing: they are cooled to allow annealing (hydrogen bonding) of short, single-stranded DNA primers complementary to sequences on opposite strands at each end of the target sequence Extension: A heat stable DNA polymerase extends the primers in the 5' to 3' direction.

usefulness of gene cloning

amplifies a particular gene and produces a protein product

agriculture

commonly used Ti plasmid which integrates a segment of its DNA into the chromosomal DNA of its host plant cells.

restriction fragment analysis

detects differences in DNA DNA fragments produced by restriction enzyme digestion (cutting) of a DNA molecule are separated by gel electrophoresis. When mixture of restriction fragments undergo electrophoresis, it yields a band pattern that looks like the starting molecule and the restriction enzyme used.

genetic engineering

direct manipulation of genes for practical purposes

DNA microarray assays

identification of genes in larger samples Isolate the mRNA Make cDNA by reverse transcription, using fluorescently labeled nucleotides. Apply the cDNA mixture to a microarray, a microscope side on which copies of single-stranded DNA fragments from the organism's genes are fixed, with a different gene in each spot. The cDNA hybridizes ith any complementary DNA on the microarray. When microarray is scanned for fluorescence, it will show a gene expressed in the sample.

techniques used for determining gene function

in vitro mutagenesis (disable a gene and look for the effects) RNAi (engineer a RNA fragment to block the gene)

gene cloning steps

isolate DNA insertion in vector Reinfect cells Clone cells Identify colonies wth recombinant plasmids

biotechnology

manipulation of organisms or their components to make useful products

environmental cleanup

microorganisms are genetically engineered to extract minerals from the environment or degrade various types of toxic waste materials.

concerns over GMOs

no labeling >50% of corn and soy have GMO little research done on environmental impact small farmers forced tobuy expensive seeds

cloning vector

original plasmid

How do restriction enzymes make recombinant DNA

restriction enzyme recognizes the restriction site and makes staggered cuts in the sugar-phosphate backbones within the sequence producing fragments with sticky ends. DNA fragment from different molecule cut by the same restriction enzyme is added. Any fragments with complementary sticky ends can base-pair, including the two original fragments. DNA ligase seals the strands. If fragments come from different DNA molecules, the ligated product is recombinant DNA

restriction endonucleases

restriction enzymes cut DNA at specific sites (restriction sites)

restriction fragments

resulting pieces of DNA

single nucleotide polymorphism

single base-pair variations can help to identify disease alleles

sticky end

single stranded ends of restriction fragments

plasmid

small circular DNA molecules that replicate separately from the bacterial chromosome

bacterial artificial chromosome

A large plasmid that acts as a bacterial chromosome and can carry inserts of 100,000 to 300,000 base pairs

Genomic library

Complete set of plasmid containing cell clones, each carrying copies of a particular segment from the initial genome

First step in gene cloning

Isolate DNA obtain vector Plasmid contains only one copy of the restriction vsite recognized by the restiction enzyme

nucleic acid hybridization

Nucleic acid probe is used to identify clones/detect a specific DNA sequence Each probe is labeled with a radioactive isotope or a fluorescent tag for detection. Cells from each well, representing oneclone, are transferred to a defined spot on a special nylon membrane. The nylon membrane is treated to break open the cells and denature their DNA; the resulting single-stranded DNA moelcules stick to the membrane. The membrane is then incubated in a solution of radioactive probe molecules complementary to the gene of interest. Because the DNA immobilized on the membrane is single stranded, the single-stranded probe can base pair with any complementary DNA on the membrane. The membrane is laid under photographic film, allowing any radioactive areas to expose the film. The film shows locations of the DNA membrane that has hybridized to the probe. Each hybridized location can be traced back to the original well containing the bacterial clone that holds the gene of interest.

gene cloning

Production of multiple copies of a single gene

complementary DNA (cDNA)

a double stranded DNA molecule made in vitro using mRNA as a template. The enzyme reverse transcriptase is used in vitro to make a single-stranded DNA using the mRNA as a template. The 3' end of the mRNA has a stretch of adenine ribonucleotides called poly-A-tail. This allows use of deT's (deoxyribonucleotides) as a primer for the reverse transcriptase. mRNA goes through enzymatic degradation A second DNA strand, complementary to the first is synthesized by DNA polymerase. Because the mRNA only contains exons, the resulting double-stranded cDNA carries the complete coding sequence of the gene but no introns. Used to study specific proteins or sets of genes expressed in particular cell types

nucleic acid probe

a labeled single-stranded nucleic acid molecule used to locate a specific nucleotide sequence in a nucleic acid sample. Moleucle of the rpobe hydrogen bodn to the complementary sequence where it occurs; radioactive, fluorescent, or other labeling of the probe allows its location to be detected.

restriction fragment length polymorphism (RFLP)

a single nucleotide polymorphism that exists in the restriction site for a particular enzyme, making the ste unrecognizable by that enzyme and changing the lengths of the restriction fragments formed by digestion with that enzyme. inherited differences in non-coding and coding fragments used in genetic mapping

expression vectors

active bacterial promoters

gene therapy

add good allele to patients cells and reinject cells not effective

cloning animals

adult cells are not totipotent Nuclear transplantation: remove nucleus of an unfertilized/fertilized egg and replace it with the nucleus of a differentiated cell such as reproductive cloning

cloning plants

adult cells can dedifferentiate and give rise to all cell types (totipotent)

southern blotting

electrophoresis and nucleic acid hybridization specific to one gene Used to detect specific nucleotide sequences within a complex DNA sample and compare restriction fragments produced from different samples of genomic DNA Preparation of restriction fragments: Each DNA sample is mixed withthe same restriction enzyme. Digestion of each sample yields a mixture of thousands of restriction fragments. Gel eletrophoresis: The restriction fragments in each sample are separated by electrophoresis, forming a characteristic pattern of bands. DNA transfer (blotting): DNA is denatured and transferred to a nitrocellulose membrane. This produces a blot with a pattern of DNA bands exactly like that of the gel. Hybridization with labeled probe: The nitrocellulose blot is exposed to a solution containing a probe labeled radioactively/fluorescent. Probe molecules attach by base-pairing to any restriction fragments containing a part of the gene of interest. Probe detection: A sheet of photographic film is laid over the blot. The radioactivity in the bound probe exposes the film to form an image corresponding to those bands containing DNA that base-paired with the probe.

storing cloned genes

foreign genome is cut with restriction enzymes into either small fragments or large fragments. If small plamsmid, they become genomic library If big plasmid, BAC Both types are stored in a multiwell plastic plate. Each clone occupies one well.

genome

genetic material of an organism or virus

forensics

genetic profile (an individual's unique set of genetic markers, detected most often by PCR, previously by electrophoresis and nucleic acid probes) short tandem repeats (STR) (simple DNA sequence used to prepare genetic profiles)

pharmaceuticals

human proteins grown in bacteria or animals (transgenic of genetically modified organisms) products produced by genetic engineering (insulin for diabetics, human growth hormone for children born with darfism, TPA to dissolve blood clots)

clone the cells

place bacteria on medium containing ampicillin. Only cells with a plasmid will reproduce because only they have the gene that is resistant to ampillin. Each reproducing bacteria forms a clone of cells.

What are the 2 different types of vectors commonly used in recombinant DNA techniques

plasmids virus

identify colonies with recombinant plasmids

presence of Xgal in medium allows us to distinguish colonies with recombinanat plasmids from those with non recombinant plasmids. Colonies containing nonrecombinant plamids have the lacz gene intact and will produce functional B-galactosidase. These colonies will be blue because the enzyme hydrolyses the X-gal in the medium, foming blue product.

disease diagnosis

recognize disease alleles before patient is symptomatic